The dmsEFABGH operon encodes an essential and modular electron transfer pathway for extracellular iodate reduction by Shewanella oneidensis MR-1

ABSTRACT Extracellular iodate reduction by Shewanella spp. contributes to iodide generation in the biogeochemical cycling of iodine. However, there is a disagreement on whether Shewanella spp. use different extracellular electron transfer pathways with dependence on electron donors in iodate reduction. In this study, a series of gene deletion mutants of Shewanella oneidensis MR-1 were created to investigate the roles of dmsEFABGH, mtrCAB, and so4357–so4362 operons in iodate reduction. The iodate-reducing activity of the mutants was tested with lactate, formate, and H2 as the sole electron donors, respectively. In the absence of single-dms gene, iodate reduction efficiency of the mutants was only 12.9%–84.0% with lactate at 24 hours, 22.1%–85.9% with formate at 20 hours, and 19.6%–57.7% with H2 at 42 hours in comparison to complete reduction by the wild type. Progressive inhibition of iodate reduction was observed when the dms homolog from the so4357–so4362 operon was deleted in the single-dms gene mutants. This result revealed complementation of dmsEFABGH by so4357–so4362 at the single-gene level, indicating modularity of the extracellular electron transfer pathway encoded by dmsEFABGH operon. Under the conditions of all electron donors, significant inhibition of iodate reduction and accumulation of H2O2 were detected for ΔmtrCAB. Collectively, these results demonstrated that the dmsEFABGH operon encodes an essential and modular iodate-reducing pathway without electron donor dependence in S. oneidensis MR-1. The mtrCAB operon was involved in H2O2 elimination with all electron donors. The findings in this study improved the understanding of molecular mechanisms underlying extracellular iodate reduction. IMPORTANCE Iodine is an essential trace element for human and animals. Recent studies revealed the contribution of microbial extracellular reduction of iodate in biogeochemical cycling of iodine. Multiple reduced substances can be utilized by microorganisms as energy source for iodate reduction. However, varied electron transfer pathways were proposed for iodate reduction with different electron donors in the model strain Shewanella oneidensis MR-1. Here, through a series of gene deletion and iodate reduction experiments, we discovered that the dmsEFABGH operon was essential for iodate reduction with at least three electron donors, including lactate, formate, and H2. The so4357–so4362 operon was first demonstrated to be capable of complementing the function of dmsEFABGH at single-gene level.

gene deletions and test them with three different electron donors to better understand their role in iodate reduction.Overall the results are clear, and while mostly confirmatory in nature, do help us better understand iodate reduction in Shewanella.Two major comments are below, along with some minor comments for the author's consideration.The experiments seem to be single biological replicates with three measurements.From the figure legends: "The values reported are the means and standard deviations of triplicate measurements."These should be biological replicates to specifically address biological variation rather than technical variation in measurement of iodate.Line 250 reports final growth yields also seem to indicate n=1, as no standard deviation for these values are presented.Biological replicates would also help validate the results of the double mutant work shown in Figure 3. Reviewer 2 raised a question (point 8) regarding negative controls for iodate reduction, specifically looking for a control where electron donor was omitted.This request is especially important considering that the anaerobic work was done primarily in a Coy anaerobic chamber.While the authors claim this is filled with 'pure nitrogen gas' this cannot be the case.To scrub oxygen from the system, Coy chambers require hydrogen gas.S. oneidensis will robustly use hydrogen as an electron donor, which may confuse the results here.Considering that the authors state that the electron donor dependence of iodate reduction was one of the major aims of this work, it is strange that controls lacking electron donor are missing.The authors need to do a better job describing their experimental setup such that someone who is interested in the results could repeat them.I don't quite understand how anaerobic tubes were flushed with nitrogen gas while also inside a Coy chamber.
Line 138 -why is this 'novel'?Isn't it simply modified?Please describe why the modification was made here.1 Growth curves in supplemental should be plotted on semi-log scale to accurately depict growth features.See https://schaechter.asmblog.org/schaechter/2018/07/why-you-must-plot-your-growth-data-on-semi-log-graph-paper.htmlSupplemental figures should be on individual pages with their accompanying figure legends.

Reviewer #3 (Comments for the Author):
This study focuses on the microbially-mediated reduction mechanism of iodate.The authors demonstrated that only dmsEFABGH was required for direct reduction of iodate without any dependence on electron donors.Also, the so4357-so4362 gene cluster of Shewanella oneidensis MR-1 was first verified to be active in iodate reduction at single-gene level.In general, this study is straightforward and the methods used are robust.The results of this study are reliable and show new findings in the field.
Detailed comments: Line 48, microorganism-strain Line 51, The sentence "These observations ---natural environments" is not supported and should be deleted.The importance of so4357-4362 function should be added.Line 69, non-biotic-abiotic Line 235, the vector itself---the empty vector Line 246, growth---incubation Line 295, the word "obvious" is conversational Line 332, the sentence "Therefore, in addition to DmsE, some unknown periplasmic electron carriers were involved in electron transfer to DmsB" should be placed in the former paragraph (line308-318).Line 341, To be consistent with gene sequences in the cluster, mtrABC should be mtrCAB.Line 367, lag---absence Line 369-392, Figure 5 is a concept graphic and should be described independently.The function of DmsGH should be indicated or described in text.

Response to Reviewers Comments
Reviewer #1: The work here focuses on better illuminating the role of the dms gene cluster in iodate reduction by Shewanella oneidensis.There have been a few papers published recently on this topic, with some discrepancies.The authors generate a series of single gene deletions and test them with three different electron donors to better understand their role in iodate reduction.Overall, the results are clear, and while mostly confirmatory in nature, do help us better understand iodate reduction in Shewanella.Two major comments are below, along with some minor comments for the author's consideration.

Q1:
The experiments seem to be single biological replicates with three measurements.Q2: Reviewer 2 raised a question (point 8) regarding negative controls for iodate reduction, specifically looking for a control where electron donor was omitted.This request is especially important considering that the anaerobic work was done primarily in a Coy anaerobic chamber.While the authors claim this is filled with 'pure nitrogen gas' this cannot be the case.To scrub oxygen from the system, Coy chambers require hydrogen gas.S. oneidensis will robustly use hydrogen as an electron donor, which may confuse the results here.Considering that the authors state that the electron donor dependence of iodate reduction was one of the major aims of this work, it is strange that controls lacking electron donor are missing.The authors need to do a better job describing their experimental setup such that someone who is interested in the results could repeat them.I don't quite understand how anaerobic tubes were flushed with nitrogen gas while also inside a Coy chamber.

Response:
As suggested by the reviewer, we supplemented a control experiment where electron donors were omitted.The procedures of medium preparation and bacterial collection and inoculation were the same as previous experiments.IO 3 − reduction was severely inhibited for both wild-type and mutant strains (Figure S1).Although Shewanella oneidensis MR-1 may utilize endogenous electron donors, only 21.4% of added IO 3 − was reduced by the wild-type strain and less IO 3 − was reduced by the mutants.However, 93.8% of added IO 3 − was reduced by the wild type with exogenous lactate as the sole electron donor.Therefore, IO 3 − reduction in previous experiments should be ascribed to the added electron donors but not H 2 contamination.
We have added this figure and corresponding descriptions in the methods and results of the text.In our study, we replaced the mixed gases (N 2 : CO 2 : H 2 = 95.5 : 3 : 1.5) in the chamber with pure nitrogen gas to eliminate the contamination of hydrogen gas used for maintaining anaerobic condition of the Coy chamber.Moreover, bacterial inoculation was performed with disposable syringe and there is no need to open the rubber stopper of anaerobic tubes and bottles.We have added these details in the Materials and methods section of the text, including "Afterward, the cells were inoculated in anaerobic tubes with disposable syringe" (Line 155) and "During bacterial collection, the mixed gases (N 2 : CO 2 : H 2 = 95.5 : 3 : 1.5) in the chamber was replaced by pure nitrogen gas".(Line 161) To eliminate dissolved oxygen, prepared medium was boiled and purged for 20 min with pure nitrogen gas.These procedures were performed outside the Coy chamber.In the revised version, we have specified the procedures that need to be implemented inside the Coy chamber as "Bacterial washing and inoculation procedures were performed in an anaerobic chamber (Coy Laboratory Products, USA)".(Line 159-160) We apologize for any misinterpretation.
Q3. Line 138 -why is this 'novel'?Isn't it simply modified?Please describe why the modification was made here.

Response:
We have deleted the word 'novel'.The revised sentence is "A suicide vector pDS3.2 was constructed by inserting a short sequence with additional SbfI and AvrII sites in plasmid pDS3.0(37)" (Line 119-120).Compared to pDS3.0, the pDS3.2vector provides more restriction sites for construction of recombinant plasmids.

Q4. Line 146 -what is the length of sequence amplified?
Response: The length of each target sequence was added in Table S1.

Q5. Line 154 -optimized how?
Response: The description "optimized ribosomal binding sites" was used to indicate that these sites have been shown to be effective in Shewanella oneidensis MR-1.We have modified the sentence as "To complement the mutants, the coding sequence of each gene was amplified by using primers with ribosomal binding sites that have been shown to be effective in S. oneidensis MR-1 (40,41)" (Line 134-136).
Q6. Line 169 -why are you growing anaerobic cultures with excess electron donor?I don't think you need to re-do these experiments, but was there a rationale for only providing half of the required fumarate?
Response: We appreciate this reminder.The ratio between the electron donor lactate and the electron acceptor fumarate was not accurately calculated.Considering that a proportion of the electrons released from lactate oxidation will be consumed in anabolism, we usually add excess lactate in anaerobic cultivation of Shewanella spp..

From
Figure S2 Growth of the wild type and dmsEFABGH mutants with DMSO as the sole terminal electron acceptor.(A) Growth over 26 hours.(B) Optical density at 22-hour.WT, S. oneidensis MR-1.(A) Growth over 26 hours.(B) Optical density at 22-hour.WT, S. oneidensis MR-1.The values reported are the means and standard deviations of triplicate experiments.For points without error bar, the error was smaller than the symbol.Asterisks, significance levels of difference between the mutants and WT, 0.01 < P < 0.05 (*), 0.001 < P < 0.01 (**), P < 0.001 (***).

Figure
Figure S1   − reduction by S. oneidensis MR-1 strains without electron donors.(A) Percentage of added IO 3 − over 36 hours of reduction and (B) Efficiency of IO 3 − reduction at 36-hour.WT, S. oneidensis MR-1.One hundred percent of added IO 3 − was equal to 250 μM.The values reported are the means and standard deviations of triplicate experiments.For points without error bar, the error was smaller than the symbol.Asterisks, significance levels of difference between the mutants and WT, 0.01 < P < 0.05 (*), 0.001 < P < 0.01 (**), P < 0.001 (***).